ABSTRACT Human papillomavirus (HPV) infection is a global threat to public health, but infection and replication remain poorly understood, thus hindering the development of antivirals for cancer prevention and treatment. The goals of this proposal are two-fold. First, we define the genetic role of the Fanconi anemia pathway in suppressing epidermal susceptibility to HPV infection and replication. Second, we will test clinically relevant inhibitors of ganglioside biosynthesis and signaling (Aim 1), and define new targets (Aim 2), to prevent or attenuate such susceptibility. The HPV life cycle takes place in human epidermis, and is intricately linked to the integrity of this stratified tissue and the differentiation of keratinocytes. There are two basic categories of keratinocytes ? epidermal stem and progenitor cells (ESPCs) located in the basal cell layer, and differentiated progeny located in more superficial layers. For the viral life cycle to begin, HPV must infect ESPCs. Access to these basal cells requires a temporary breakdown in epidermal integrity. Infected ESPCs then migrate to the surface, differentiating en route. Viral genome amplification is triggered in a poorly characterized subset of terminally differentiated cells, followed by encapsidation and release of infectious progeny. Our recent epidemiological studies of the inherited genome instability disorder Fanconi anemia (FA) demonstrated that FA patients have a significantly increased risk of HPV positivity, suggesting that FA pathway loss of function may increase susceptibility to HPV infection and/or proclivity for amplification. Our published and preliminary data indicate that FA pathway deficiency stimulates the HPV life cycle at two critical stages: initial infection and late amplification. In the absence of HPV, FA pathway deficiency diminished keratinocyte adhesion, and accelerated skin blistering ? suggesting structural impairment of the host tissue, which could facilitate HPV infection. This will be tested in patient-derived FA-inducible and conventional systems using electron microscopy, molecular investigation of mechanisms focused on lipid metabolism, and studies of HPV infectivity. Available ganglioside biosynthesis and Rac1 inhibitors will be tested for their ability to prevent initial HPV infection via restoration of epidermal integrity. In the presence of HPV, FA pathway deficiency triggered excessive and ectopic viral genome amplification ? suggesting that the intact FA pathway suppresses HPV replication and progeny production. This hypothesis will be tested by generating an HPV+ replication system conditional for FA, and by single-cell RNA sequencing that will identify transcriptomic distinctions between HPV-replicating and -nonreplicating cells, in the presence and absence of a functional FA pathway (Aim 2). Candidate regulators will be validated and mapped in 3D epidermis, and putative effectors targeted to attenuate HPV replication in the FA hyper-permissive (and normal) human host. Together, we take the first required step towards discovering new targets and chemopreventive agents that endow human epidermis with maximal integrity and resistance to HPV infection and amplification.